PROJECT SUMMARY Proper organization of microtubules (MTs) is critical for development and function of neural circuits. MTs form the tracks on which the molecular motors kinesin and dynein transport cargo to specific cell locations. The inherent plus end/minus end polarity of MTs directs motor transport. In axons MTs are arranged with their plus ends distal to the cell body. MT polarity must be established during initial axon formation, and also during axon branching events. The mechanisms regulating MT polarity in axons and branches are poorly understood, but a body of evidence suggests that molecular motors are crucial to MT polarity. Kinesin and dynein contribute to MT polarity and are hypothesized to act by transport of MTs. However, visualizing MT polymer transport is challenging and has only been done in vitro or cultured neurons. Our lab showed previously that Clstn1, a kinesin-1 adaptor, regulates axon branching in sensory neurons. Our preliminary data show that Clstn1 is also required for MT polarity in axons. How a kinesin adaptor, known primarily to mediate cargo transport, also regulates MT polarity is unknown. My goal is to reveal the underlying mechanisms. Clstn1 is known to bind and activate KLC, which activates KHC and kinesin motor activity, and inhibits KHC tail binding to MTs. I hypothesize that Clstn1 activation of KLC prevents excessive MT crosslinking by KHC tail-MT binding. In the absence of Clstn1, this crosslinking may oppose dynein?s ability to remove misoriented MTs from the axon, leading to an increase in mispolarized MTs. I will use high-speed in vivo 4D imaging approaches to test these hypotheses. In Aim 1, I will first test if activation of KLC using a Clstn1 W-acidic domain peptide is sufficient to rescue MT polarity in Clstn1-/- mutants. Second, I will use two approaches to disrupt the binding of the KHC tail to MTs to reduce KHC crosslinking to MTs, and test for rescue of MT polarity Clstn1-/- mutants. Finally, I will test the roles of KLC phosphorylation and proteolytic cleavage of Clstn1 in MT polarity. In Aim 2 I propose to use advanced imaging technologies in collaboration with the Laboratory of Optical and Computational Instrumentation to image MT dynamics in vivo. I will label MT polymers and plus/minus ends with multiple fluorophores, and use fluorescence recovery after photobleaching to determine whether MT transport contributes to polarity in vivo. I will test whether Clstn1 loss affects MT transport, and thereby influences polarity. I will also test the hypothesis that Clstn1 functions to organize MT polarity during axon branching by imaging MT dynamics at branch points. These experiments will enhance our understanding of the cellular and molecular mechanisms that establish axon MT polarity.